CircDCLRE1C Regulated Lipopolysaccharide-Induced Inflammatory Response and Apoptosis by Regulating miR-214b-3p/STAT3 Pathway in Macrophages

Abstract

The immune cell inflammation response is closely related to the occurrence of disease, and much evidence has shown that circular RNAs (circRNAs) play vital roles in the occurrence of disease. However, the biological function and regulatory mechanisms of circRNAs in the immune cell inflammation response remain poorly understood. In this study, we constructed an inflammatory model using lipopolysaccharide (LPS)-stimulated chicken macrophage lines (also known as HD11) to verify the function and mechanism of the novel circDCLRE1C (ID: gga_circ_0001674), which was significantly upregulated in spleen tissues infected by coccidia and the macrophage cells exposed to LPS. The results showed that circDCLRE1C aggravated LPS-induced inflammation and apoptosis in HD11 cells. Systemically, circDCLRE1C acted as a sponge for miR-214b-3p binding sites thereby regulating the expression of STAT3. The overexpression of miR-214b-3p rescued the pro-inflammatory effect of circDCLRE1C in HD11 cells stimulated with LPS, and rescued the high expression of STAT3. In conclusion, our study showed that circDCLRE1C could aggravate LPS-induced inflammation and apoptosis through competitive adsorption of miR-214b-3p, thereby increasing the expression of STAT3.

Keywords: STAT3; apoptosis; circular RNA; inflammation; macrophage; miR-214b-3p.

Figure 1

Characteristics of circDCLRE1C: (A) The schematic representation of circDCLRE1C’s formation. The back-spliced junction (BSJ) of circDCLRE1C was validated by PCR using a divergent primer, followed by Sanger sequencing. (B,C) Northern blot analysis for the detection of circDCLRE1C expression in HD11 cells treated with or without RNase R, respectively; divergent primers amplified circDCLRE1C from complementary DNA (cDNA), but not from genomic DNA (gDNA). (D) The relative expression levels of circDCLRE1C in the nucleus and cytoplasm were detected by qRT-PCR. (E,F) The expression of cytokines (IL-6, TNF-α, and IFN-γ) and circDCLRE1C, respectively, in the spleens of non-infected and infected chickens, was detected by qRT-PCR; n = 4. (G) qRT-PCR analysis of the expression levels of cytokines (IL-6, TNF-α, and IFN-γ) and circDCLRE1C, respectively, induced by LPS stimulation in HD11 cells. Data are presented as means ± SD (n = 3 biologically independent samples). ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 2

CircDCLRE1C aggravated LPS-induced inflammation and apoptosis in HD11 cells: (A,B) ELISA and qRT-PCR assay showing the expression levels of IL-6, TNF-α, and IFN-γ in HD11 cells with circDCLRE1C knockdown or overexpression. (C,D) Cell apoptosis measured with Annexin V–FITC/PI apoptosis detection kit via flow cytometry assay. (E) The mRNA levels of STAT3 in HD11 cells with circDCLRE1C overexpression and knockdown. Data are presented as means ± SD (n = 3 biologically independent samples). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 3

CircDCLRE1C could serve as a sponge for miR-214b-3p to regulate the expression of STAT3: (A,C) The potential binding site sequence (highlighted in red) of miR-214b-3p on circDCLRE1C or STAT3, and the model of interaction between miR-214b-3p and circDCLRE1C or STAT3, modeled by RNAhybrid software. (B,D) Dual-luciferase assay verifying the binding relationship between miR-214b-3p and circDCLRE1C or STAT3, respectively. (E) The mRNA level of STAT3 in HD11 cells with circDCLRE1C overexpression and knockdown, as measured by qRT-PCR. (F) The mRNA level of STAT3 in HD11 cells with miR-214b-3p mimics and inhibitors. (G) The mRNA level of miR-214b-3p in HD11 cells with circDCLRE1C overexpression and knockdown. (H) The increased expression of STAT3 in HD11 cells with circDCLRE1C overexpression could be rescued by mi-214b-3p. (I,J) CircDCLRE1C was pulled down by the biotin-miR-214b-3p probe, as detected by gel electrophoresis and qRT-PCR. The relative levels were normalized to the input. Data are presented as means ± SD (n = 3 biologically independent samples). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 4

miR-214b-3p alleviated LPS-induced inflammation and apoptosis in HD11 cells: (A,B) The expression levels of miR-214b-3p were reduced in coccidia-infected samples and the LPS-induced HD11 inflammatory model, as detected by qRT-PCR. (C,D) The expression levels of IL-6, TNF-α, and IFN-γ in LPS-stimulated HD11 cells with miR-214b-3p mimics and inhibitors, as detected by qRT-PCR and ELISA. (E,F) Cell apoptosis was detected with Annexin V–FITC/PI double-staining via flow cytometry assay. (G) The mRNA levels of STAT3 in HD11 cells with miR-214b-3p mimics and inhibitors. Data are presented as means ± SD (n = 3 biologically independent samples). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 5

STAT3 aggravated LPS-induced inflammation and apoptosis in HD11 cells: (A,B) The mRNA expression level of STAT3 was increased in coccidia-infected samples and LPS-induced inflammation in HD11 cells, as detected by qRT-PCR. (C,D) The expression levels of IL-6, TNF-α, and IFN-γ in LPS-stimulated HD11 cells with STAT3 overexpression and knockdown were detected by qRT-PCR and ELISA. (E,F) Cell apoptosis was detected with Annexin V–FITC/PI double-staining via flow cytometry assay. Data are presented as means ± SD (n = 3 biologically independent samples). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 6

MiR-214b-3p mimics partly reversed the effects of circDCLRE1C in the inflammation responses: (A) MiR-214b-3p mimics could rescue the increase in the STAT3 mRNA expression caused by overexpression of circDCLRE1C in HD11 cells stimulated with LPS. (B) High expression levels of IL-6, TNF-α, and IFN-γ induced by overexpression of circDCLRE1C were rescued by co-transfection with circDCLRE1C and miR-214b-3p mimics in the inflammatory response. (C,D) The Ov-circDCLRE1C-induced increase in cell apoptosis was reversed by co-transfection with ov-circDCLRE1C and miR-214b-3p mimics. Data are presented as means ± SD (n = 3 biologically independent samples). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test). (E) Graphical abstract: graphical diagram of circDCLRE1C promoting LPS-induced inflammation and apoptosis by sponging miR-214b-3p and regulating STAT3 expression.